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Alejandro Conde-Perez1,2,3 & Lionel Larue*1,2,3 Institut Curie, Developmental Genetics of Melanocytes, Bat. 110, 91405, Orsay, France CNRS, UMR3347 Bat. 110, 91405, Orsay Cedex, France 3 INSERM, U1021 Bat. 110, 91405, Orsay Cedex, France *Author for correspondence: Tel.: +33 1 69 86 71 07 n Fax: +33 1 69 86 71 09 n [email protected] 1 2

The PI3K–PTEN–AKT signaling pathway is involved in various cellular activities, including proliferation, migration, cell growth, cell survival and differentiation during adult homeostasis as well as in tumorigenesis. It has been suggested that the constitutive activation of PI3K/AKT signaling with concurrent loss of function of the tumor suppressor molecule PTEN contributes to cancer formation. Members of the PI3K–PTEN–AKT pathway, including these proteins and mTOR, are altered in melanoma tumors and cell lines. A hallmark of activation of the pathway is the loss of function of PTEN. Indeed, loss of heterozygosity of PTEN has been observed in approximately 30% of human melanomas, implicating this signaling pathway in this cancer. PI3K signaling activation, via loss of PTEN function, can inhibit proapoptotic genes such as the FoxO family of transcription factors, while inducing cell growth- and cell survival-related elements such as p70S6K and AKT. Determining how the PI3K–PTEN–AKT signaling pathway, alone or in cooperation with other pathways, orchestrates the induction of target genes involved in a diverse range of activities is a major challenge in research into melanoma initiation and progression. Moreover, the acquisition of basic knowledge will help patient management with appropriate therapies that are already, or will shortly be, on the market.

Malignant melanoma is the most highly aggressive type of skin cancer, and the incidence of its diagnosis in westernized countries is increasing. Identification of the molecular mechanisms of melanoma formation (melanomagenesis) is imperative, as this cancer has a high propensity to metastasize and in many cases is resistant to currently available treatment. There has been extensive research undertaken to dissect and describe the key genetic and epigenetic alterations that result in aberrant signaling and subsequent malignancy. Several regulatory pathways play important roles in melanomagenesis including, but not limited to, MAPK, WNT and PI3K (Figure 1) . Although there are undoubtedly cooperative effects between these pathways, this issue is beyond the scope of this review. Constitutive activation of PI3K, via gain-of-function mutations in the PIK3CA gene encoding the class I PI3K catalytic subunit p110a, are observed in a variety of malignancies, from glioblastomas to lung cancers [1,2] , but only infrequently in melanoma. However, mutation, epigenetic and deletion events involving the tumor suppressor PTEN may be present in as many as 40–50% of sporadic melanomas [3] . Loss of PTEN function is a major contributing factor to increased signaling downstream from PI3K/AKT. A number of PI3K–PTEN–AKT cascade components have been identified, but their functions and regulation in the melanocyte lineage are still poorly 10.2217/FON.12.106 © 2012 Future Medicine Ltd

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PTEN and melanomagenesis

understood. This is partly because most of the functional studies addressing PTEN function have used carcinoma, prostate and breast cancer models. Dissection of the PI3K–PTEN–AKT axis in melanoma should provide further insight into the melanocyte-specific molecular and cellular events involved in the initiation and/or progression of melanocytes towards melanoma. Melanomagenesis

Melanomagenesis is a multistep process that develops through a series of stages: n Common acquired nevi and dysplastic nevi; Radial growth phase melanoma;

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Vertical growth phase melanoma;

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Metastatic melanoma [4,5] . In a schematic way, considering cellular mechanisms, melanomagenesis may be defined as the initiation and progression of melanoma, in which initiation includes proliferation and bypassing senescence, and progression includes invasion and metastasis [6] . Invasion results from a combination of mechanisms: n

Pseudoepithelial-to-mesenchymal transition (melanocytes are not epithelial cells);

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Keywords n

Loss of cell–cell adhesion;

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catenin n LOH n MAPK melanoblasts n PI3K

Loss of cell–matrix adhesion;

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HGF

IGF

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FGF *

FRZ

RTK * RAS *

* RAF

PI3K

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AKT

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ERK

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* NF-κB

DSH

GSK3β

NF-κB *

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* PTEN ∆

M-MITF

β-cat ∆

LEF p16

Figure 1. Oversimplified signaling pathways associated with PTEN. Three main signaling pathways (PI3K, MAPK and WNT/b‑catenin) are connected with PTEN. Some downstream targets are induced in normal conditions. Associated proteins can be mutated. Mutations are depicted as point mutations (*) or deletions (D).

Chemo-attraction/repulsion;

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Migration.

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Metastasis formation requires intravasation, extravasation, implantation at sites and subsequent angiogenesis. Typically, benign tumors remain ‘stuck’ at or before radial growth phase, and are proliferative but noninvasive. However, the acquisition of further genetic aberrations or epigenetic modifications often leads to a bypass of the senescence barrier and a phenotypic switch from proliferative to invasive. This is accompanied, during the later stages of the disease, by colonization of distal organs, including lung, liver and brain. Metastatic melanoma ultimately becomes highly resistant to all current forms of therapy. General clinical information

The most basic mode of early detection/identification, within the first stage, utilizes the ABCD guideline system [7] : Asymmetry;

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Border irregularity;

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Color;

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Diameter.

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This approach is widely acknowledged to be insufficient and misleading, as irregularity and heterogeneity varies within samples and not all relevant growths are necessarily identified [8,9] . Accurate diagnosis often requires a skin biopsy, which still remains the quickest method of detection [10] . The Breslow index is a prognostic factor for melanoma based on measuring the depth of tumor [11] . Monitoring the Breslow thickness in patients through time can be informative: higher values of the index are positively correlated with poor prognosis and can be used as an indicator for surgical removal. Currently, clinical evaluations for loss of PTEN involve a combination of sequencing and immunohistochemical techniques, with the latter being more effective in endometrial carcinomas [12] . These techniques can be readily applied to melanoma samples. Current therapy for melanoma

Although major improvements have been made in decoding the mechanisms involved in melanomag enesis, translation into clinical applications has unfortunately not been so effective. Over the last decade, melanoma research has focused on identifying possible therapeutic targets downstream of the R AS future science group

PTEN & melanomagenesis

signaling cascade and recently PI3K–PTEN– AKT, and seeking potential inhibitors. Two molecules, vemurafenib (PLX4032) and dabrafenib (GSK2118436), have been identified as inhibitors targeting BRAF mutations, specifically the V600E mutation. BRAF inhibitors have shown great promise in the clinic: 80% of patients with tumors associated with a V600E mutation in BRAF respond to the treatment. Unfortunately, resistance emerges approximately 6–9 months after the start of treatment with these agents. This resistance may be due to compensation by the MAPK and/or PI3K pathways. It has been shown that PTEN loss confers BRAF inhibitor resistance in melanoma cells [13] . In addition, resistance may also arise via a switch in signaling from BRAF to the CRAF isoform and subsequent hyperactivation of MEK–ERK [14] . In accordance, tumor samples obtained from melanoma patients treated with vemurafenib carry an activating mutation in MEK1 (C121S), which was absent from pretreated tumors; this mutation results in increased kinase activity and strong resistance to the inhibitor [15] . Similarly, mutation of MEK1 (K59del) decreases sensitivity to dabrafenib treatment in A375 human melanoma cells [16] . Thus, resistance to currently available BRAF inhibitors appears to result from a signaling switch between RAF isoforms and MEK1 mutations. Another therapeutic agent currently in use is the anti-CTLA4 antibody ipilimumab, with 10–20% of patients responding positively to the therapy. CKIT activity inhibitors may also be beneficial [17,18] . Inhibitors targeting functional components of the PI3K–PTEN–AKT pathway are available. The best characterized is rapamycin, which inhibits the mTORC1 complex and, at high concentrations, mTORC2. The mTOR complex is an attractive target for therapeutic exploitation as it regulates cell growth and survival. Treatment with rapamycin in in vitro cellular models leads to increased apoptosis, and decreased metastatic potential [19] . Currently, efforts are being made to develop isoform-specific mTOR inhibitors, in order to increase effectiveness while decreasing off-target effects [20] . Recent evidence has suggested that combinatorial therapy using BRAF inhibitors with mTOR inhibitors may be beneficial to bypass initial resistance to the BRAF inhibitor [21,22] . Various in vitro studies indicate that combinatorial therapies may provide the basis for new treatment strategies in the near future; nevertheless, the development of appropriate and effective regimens of this type for patients is still far from trivial. future science group

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Different types of melanoma

Melanoma exists in different forms. Superficial spreading melanoma accounts for approximately 40–70% of all cases of melanoma [23–26] . The most common locations are the legs of women and the backs of men, and they occur most commonly between 30 and 50 years of age. Many are barely raised from the surrounding skin and vary in color. Such melanomas evolve over 1–5 years and can be readily caught at an early stage if they are detected and removed. Nodular melanoma is the second most common subtype, and is described in 15% of melanoma cases [27] . It is vertically invasive and generally has a higher propensity to metastasize. They present as symmetrical nodules with colors ranging from blue, to brown, pink and grey (for review, see [28]). Lentigo maligna melanoma typically develops on the chronically sun-exposed skin of the head and neck [29] . Precursor lesions are termed lentigo maligna and commonly appear as irregular brownish pigmented macular lesions that persist for years. The incidence increases with age and generally peaks at between 70 and 80 years of age [30] . Acral lentiginous melanoma (ALM) is the rarest type of melanoma (~1% of malignant melanomas for the Australian population) and occurs at the same frequency in people of different phototypes [23] . It has a worse prognosis than the other types of melanoma [31–33] . Dermatological signs include dark, irregular macules, papules or nodules on the feet and, less commonly, the hands. Subungual occurrence in the fingers is uncommon, and is reported in 1–13% of all cases of ALM [34] . Histologically, ALM is characterized by asymmetric, poorly circumscribed proliferation of continuous single melanocytes at the dermal–epidermal junction [35] . The four subtypes display different histopathological features, diagnostic criteria and overall survival rates. Different genetic alterations are associated with different subtypes of the disease. The modification of PTEN may affect the development of superficial spreading melanoma, nodular melanoma and lentigo maligna melanoma, but does not seem to influence ALM [36] . Comparative genome hybridization analysis showed that chromosomal amplifications in regions 5p15, 5p13, 12q13–22 and 16q21–22 are more frequently associated with ALM and may drive tumor formation, irrespective of the status of 10q23 where PTEN is located [36] . However, this does not exclude that PTEN expression is altered in ALM. www.futuremedicine.com

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Melanomagenesis: initiation & progression

The transformation of melanocytes, which are located in the skin and produce melanin pigments, leads to malignant melanoma. Both epigenetic and genetic alterations contribute to the etiology of melanoma. This includes genes corresponding to WNT/b‑catenin ( b‑catenin and APC), MAPK (BRAF and NRAS) and PI3K (PTEN and AKT3) signaling, cell adhesion (ITGB3, ITGAV and CDH1) and cell cycle control (CDKN2A or INK4A–ARF, MYC, RB1 and TP53) [37–39] . During the initial stage of melanomagenesis, abnormal melanocytic growth is observed in the form of nevi (moles). The cause of this abnormal proliferation is probably uncontrolled cell division and/or delayed senescence. Generally, once senescence occurs, these nevi do not progress towards malignancy. Mutated and hyperactive forms of NRAS and BR AF have been shown to be associated in melanocytes with proliferation and induction of senescence, respectively [40,41] . According to this, immortalization of the cells is required for melanoma to form. However, one must not forget that melanoma may still arise from a single melanocyte when both proliferation and immortalization events occur. In this case, it is possible that the molecular event associated with immortalization may occur first without any apparent lesion. In melanocytes, the MAPK (RAS/RAF/MEK/ERK) pathway is activated by growth factors, including SCF, HGF and FGF, resulting in ERK activation and thus proliferation [6,42] . BRAF is mutated in up to 70% of benign nevi and melanomas, leading to constitutive activation of the MAPK pathway [43] . Oncogenes such as BRAF (or NRAS) are themselves incapable of transforming primary cells, or can do so only poorly, as they cause oncogene-induced senescence [40,44,45] . Mutation and/or alteration of gene expression may lead to the immortalization of melanocytes. The P16/RB and P14/P53 pathways are classically involved in the bypass of senescence. In melanoma, the P16/RB pathway is particularly important in overcoming the senescence barrier to oncogenic stress, and P53 plays a larger role in melanomagenesis during melanoma progression (for review, see [46]). Deletion, mutation and silencing of P16 are the main molecular processes associated with the bypass of senescence in melanoma. In accordance, it has been shown that P16 can be silenced by repression by the b‑catenin–TCF4 complex or by methylation of its promoter [37] . Understanding how oncogenic 1112


cells overcome this senescence barrier to hyperproliferate is one of the keys to improving our knowledge of melanoma initiation. PTEN

The gene encoding TEP1/MMAC1 was originally identified as a tumor suppressor, later on renamed PTEN due to its propensity to be mutated or lost in advanced cancers, resulting in a loss of function. It was formally proven as a tumor suppressor in vivo once its absence led to initiation of tumors. It shares significant similarity with the catalytic domain of protein phosphatases and the cytoskeletal proteins auxillin and tensin; consequently, it was classified as a dual protein and lipid phosphatase [47,48] . However, unlike other protein phosphatases, PTEN preferentially dephosphorylates phosphatidylinositol 3,4,5-trisphosphate (PIP3) at the 3´ position [49] , thereby negatively regulating the AKT signaling pathway. PTEN structure

The PTEN protein has 403 amino acids and two key functional domains: a phosphatase domain and a C2-spanning region regulating membrane stability [50] . It has an N-terminal phosphatidylinositol 3,4-bisphosphate (PIP2) binding region and a C-terminal PDZ domain regulating protein–protein interactions [51,52] . The N-terminal domain, the first 185 amino acids, contains a protein tyrosine phosphatase signature motif and shows structural similarity to the dual-specificity phosphatase VHR (F igur e 2) . However, crystallographic analysis reveals that the residues corresponding to the active site in PTEN generate a larger groove for substrate binding than is observed for VHR, such that the substrate specificity is different [50] . The active site contains a HCXXGXXR amino acid signature commonly found in protein tyrosine phosphatases and dual specificity phosphatases [50] . The C2 domain (amino acids 186–352) contains a CBR3 loop differing slightly from those in PLCd1, PKCb and cPLA2, with only one calcium-binding residue (Asp286). Nevertheless, the loop has an overall net positive charge, resembling the Ca 2+ -dependent binding sites in the C2 domains of PLCd and PKCd; these structures may be involved in membrane binding [50] . The PIP2-binding region also contributes to the protein associating with the membrane [51] . Residues 401–403 in the PDZ-binding domain have been shown to mediate binding to MAG family kinases [52] . future science group


1

15

PBM

185

Phosphatase domain

351

C2 domain

400

Reg domain

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403 AA

PDZ

Figure 2. PTEN structure. PTEN is composed of 403 AAs. It possesses five main domains or motifs. The plasma membrane-binding motif allows the binding of PTEN to PIP2. The phosphatase domain allows the dephosphorylation of PIP3 and, with lower efficiency, of proteins such as FAK. PTEN mutations found by deep sequencing (THR167ALA, CYS136TYR and TYR155CYS) map to this domain. The C2 domain allows the stabilization of PTEN to the membrane. The Reg domain, or regulatory domain, contains phosphorylable residues (Ser362, Thr366, Ser370 and Ser385) that are important for PTEN activity. PDZ allows the interaction with MAG family kinase. AA: Amino acid.

PTEN function

PTEN has been implicated in a plethora of cellular processes. A major role of PTEN is to dephosphorylate PIP3 resulting in an increase in the amount of PIP2 species and subsequently leading to a decrease in AKT signaling (reviewed in [53]). PIP2 is a major substrate of the PI3 family of kinases and its phosphorylation by PI3K, on the third hydroxyl group, results in production of the lipid second messenger, PIP3, which enables AKT binding to the membrane and subsequent phosphorylation of THR308 by PDK1 and SER473 by mTORC2 complex, leading to its activation [54,55] . This in turn leads to activation of the downstream signaling components of the PI3K–AKT cascade, ultimately modulating cellular processes including cell growth, cell size, cell cycle progression, cell survival, proliferation, growth and angiogenesis [56,57] . PTEN also has nuclear functions. In the majority of cutaneous melanomas PTEN may be found in the nucleus. Absence from the nucleus has been associated with poor prognosis [58] . The absence of PTEN from the nucleus has also been described in other cancers including colorectal cancer, pancreatic islet cell tumors and large B-cell lymphoma, leading to the view that the tumor suppressive activity of PTEN is not solely associated with its cytoplasmic functions [59–61] . Indeed, PTEN has been reported to be predominantly cytoplasmic in neoplastic tissue, although nuclear in normal tissue, suggesting the existence of a nuclear tumor-suppressing activity for this protein [62] . It is thought that nuclear PTEN is involved in cell cycle progression, chromatin stability and control of double-strand break DNA repair by interaction with the RAD51 promoter [63,64] . PTEN has been reported to interact directly with the promoter of P53, thereby increasing future science group

P53 transcript and protein levels [65] , although the role of the PTEN–P53 interaction is controversial as others have reported that PTEN inactivation leads to an increase of P53 production [66] . Nuclear PTEN is also involved in promoting APC–CDH1 complexing resulting in cellular senescence [67] . The upregulation of PTEN is also associated with an increase in energy expenditure and mitochondrial oxidative phosphorylation providing a protective effect against oncogenic transformation [68] . Regulation of PTEN

The gene for PTEN maps to the chromosomal region 10q23, and is often lost in various malignancies [49] . Loss of function of PTEN is often associated with an increase of PIP3 within the cells and subsequent activation of downstream AKT signaling. Indeed, re-expression in PTEN‑null cells leads to decreased cell proliferation and tumorigenicity [3] . PTEN activity is regulated by a multitude of processes, including post-translational acetylation by p300/CBP, which negatively affects its function [69] . Oxidative stress impairs PTEN catalytic activity by reducing cysteines 71 and 124, resulting in disulfide bonds [70–72] . NOTCH1 also regulates PTEN expression [73] . PTEN is mono- and poly-ubiquitinated by the E3 ubiquitin ligase, and NEDD4-1 has been reported to mediate its subcellular translocation and degradation, although this is still debated [74–76] . In several types of malignancy, PTEN expression is affected by miRNA 26a and 21, and the 106b‑25 miRNA cluster. Interestingly, the PTEN pseudogene PTENP1 was recently found to compete for miRNA targeting PTEN, thereby affecting the delicate balance of regulation [77] . Recently, PREX2 and MAGI2, known to interact with PTEN, were found to www.futuremedicine.com

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be mutated [78] . The presence of these mutations may affect their interactions with PTEN. Functionally, it was previously shown that PREX2 negatively regulates the phospholipid activity of PTEN. However, it was not shown whether such mutation was effectively affecting PTEN activity when PREX2 was mutated. Lastly, PTEN activity is regulated by a series of phosphorylation events driven sequentially through CK2 and GSK3b [79–81] . These regulatory effects all have consequences for cellular processes that are associated with migration, proliferation, apoptosis, metabolism and the cell cycle. Commonly associated PTEN disorders in humans

PTEN mutations and deletions have been described in many human cancers including, but not limited to, prostate cancer, breast cancer and melanoma. Germline mutations have been found to be associated with Cowden’s, Lhermitte–Duclos and Bannayan–Zonana syndromes resulting in increased susceptibility to developing breast and nonmedullary thyroid carcinoma, dysplastic gliocytoma and endometrial carcinoma [82,83] . Over 80% of patients diagnosed with Cowden’s syndrome carry mutations in the PTEN gene [84] . The most common symptom associated with Cowden’s syndrome is the presence of multiple hamartomas in various organs [83] . Patients with Lhermitte–Duclos syndrome present obstructive hydrocephalus and mass effects, characterized by focal enlargements of the cerebellar folia [85] . Similarly, patients with Bannayan–Zonana syndrome exhibit cephalic abnormalities, mainly macrocephaly, as well as vascular malformations, subcutaneous and visceral lipoma, and intestinal hamartomas [86] . The most frequently mutated site (in ~30% of cases) in the PTEN gene is exon 5, which encodes the phosphatase domain [82] . Somatic mutations have been reported in a large number of cases of endometrial carcinoma and glioblastomas, and with varying degrees of frequency in prostate, breast, ovarian and skin cancers [82] . Involvement of PTEN in melanocyte transformation

The knowledge acquired regarding the development and the establishment of the melanocyte lineage is very informative about melanoma genesis, although it only provides a deforming mirror image [87] . At the molecular level, this cumulative knowledge is certainly true for any 1114


gene/protein, including PTEN. For instance, cells move in the organism during both embryonic and cancer development. For oncologists, the movement of a ‘transformed’ cell in the organism is not spatially and temporally controlled by this cell itself, and is discordant; consequently, the term ‘invasion’ may be better adapted. For embryologists, the movement of a cell in the organism has two characteristics: it is spatially and temporally controlled and is in harmony with the other cells. As a result, the term ‘colonization’ is generally used. Despite these differences, both ‘colonization’ and ‘invasion’ result from combinations of different mechanisms: loss of cell–cell adhesion, loss of cell–matrix adhesion, matrix degradation, chemo-attraction/ repulsion and migration. The PI3K pathway is clearly involved in these different cellular mechanisms [88–91] . For instance, it has been shown that AKT regulates E-cadherin expression, therefore it is important for cell–cell adhesion [88] . Molecular oncologists often refer to invasion as observed in tests in vitro using various matrices, for example Matrigel™ (BD Biosciences, NJ, USA). Obviously this test, although very useful, only partly reflects the full invasion process in vivo, as it addresses mainly matrix degradation and chemo-attraction/repulsion in vitro. During the establishment of the melanocyte lineage, embryologists refer to migration and not to colonization. This is because the loss of cell–cell adhesion, loss of cell–matrix adhesion and matrix degradation do not seem to be important at this stage: the tissues are very ‘loose’ until the end of organogenesis (~E14.5) and become tighter during fetal development. Consequently, migration remains the main cellular mechanism for melanoblast movements until that period. PTEN in the establishment of the melanocyte lineage

The exact role of PTEN in embryonic development has not been clearly elucidated, but experiments in mice showed that it is clearly important as homozygous loss leads to embryonic lethality at the time of gastrulation. The role of PTEN in the establishment of the melanocyte lineage is also not known, but the absence of this protein does not lead to major coat color phenotype in mice [92] , and PTEN is produced in murine melanoblasts [93] . Interestingly, after backcrossing the mice more than ten-times on C57BL/6 the hyperpigmentation in the tail, pinna, paws, skin and olfactory bulb in HM mice disappeared [Puig I et al., Unpublished Data] . At this point, the involved modifier gene(s) remain unknown. future science group


PTEN is absent in a large proportion of melanomas

Various mutations in the PTEN gene have been identified in cutaneous melanoma: the frequency of their occurrence is reported to be approximately 10% for primary melanoma and 40% for melanoma cell lines [57] . Several groups have identified somatic mutations occurring at the PTEN locus in primary and metastatic melanoma with a higher frequency in metastatic samples [94,95] . Mutations can occur all throughout the PTEN coding region. The most common genetic alteration of PTEN in melanoma, associated with loss of function and melanoma development, is loss of heterozygosity [96] . Recently, three independent deep-sequencing studies were performed, two in melanoma cell lines and one on melanoma metastasis [78,97,98] . Besides the frequent chromosomal rearrangement, loss of heterozygosity of the region and the classical PTEN deletion, PTEN can present some point mutations (THR167ALA, CYS136TYR and TYR155CYS) in which phosphorylation of PTEN can be potentially affected. Role of PTEN in melanomagenesis

To elucidate the role of PTEN in melanoma initiation and progression, various in vitro and in vivo models have been established and characterized. Such models can also be used to screen drugs and new therapies. The constitutive inactivation of the Pten gene in mice leads to death at embryonic day 9.5 when homozygous. Heterozygous mice are viable and fertile but develop a large range of tumor types, except melanoma. The importance of PTEN in melanomagenesis was assessed using constitutively heterozygous mice and conditionally mutated mice for this gene. Early on, mice mutated for Pten, as heterozygous, and Ink4A/Arf, as homozygous, produced cutaneous melanoma [99] . These results provided the first evidence that PTEN was a cause of melanoma initiation. However, this mouse model was far from optimal for the study of melanoma; the penetrance was low (three out of 46), the latency period was long (~7 months) and, more importantly, many other types of tumor arose earlier (from 7 weeks of age) and more frequently (in ~75%). Mouse molecular genetic technology has allowed the generation of conditional mouse mutants for Pten using the CRE–LoxP system [6] . To generate melanocyte-specific transgenic expression, regulatory sequences of tyrosinase family genes, such as those from Tyr itself, Dct and Tyrp1 are used by many laboratories [100] . The Tyr promoter has been manipulated for future science group

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better expression in transgenic mice [101,102] . Subsequently, a combinatorial approach of TYR enhancer and Tyr promoter sequence was used to generate transgenic mouse lines enabling a melanocyte-specific expression of CRE recombinase (TYR::CRE, spatial control) [103,104] and tamoxifen-inducible expression of CRE recombinase (TYR::CREERt2, spatio-temporal control) [105,106] . The expression of CRE recombinase was also successful in the melanocyte lineage after insertion of CRE into the Dct locus, although expression is weaker than that from TYR::CRE constructs [107] . The inactivation of Pten in the melanocyte lineage, and some neural crest derivatives, using the DCT::CRE mice led to 50% lethality after birth with the remaining population having increased melanocyte counts in the skin and displaying susceptibility to carcinogen-induced melanomagenesis [108] . Even though DCT::CRE mice are not efficiently recombining floxed genes present in melanocytes, the specific inactivation of PTEN in the melanocyte lineage leads to a resistance to hair graying [108] . These results suggest that PTEN is important in the renewal of melanocytes. Several explanations can be given. In the absence of PTEN the number of melanocyte stem cells in each bulge is more important; melanocyte stem cells overcome natural senescence or transit amplifying cells divide more than normal for each cycle. It is widely accepted that NRAS activates both MAPK and PI3K pathways, but BRAF activates only the MAPK pathway. Conditional inactivation of PTEN and activation of BRAF in mice after birth has been used to evaluate synergy between the MAPK and PI3K signaling pathways. These mutant mice formed melanoma with short latency and 100% penetrance. Metastasis to distal organs, lung and lymph nodes was observed [109] . The conditional expression of a stabilized form of b‑catenin in a BRAFV600E, PTEN-null context led to the acceleration of melanoma initiation and increased metastasis [42,110] . It has also been shown that PTEN ceRNA cooperates with BRAF V600E to promote melanomagenesis, providing further evidence of the importance of PTEN regulation in melanoma formation [111] . At the molecular level, it is important to note that PTEN deficiency can cooperate with HRASV12G hyperactivation to promote murine melanomagenesis [112] . The relevance for human melanoma is questionable as HRAS mutations are found in only 1% of cases, and concomitant mutation of RAS (N, K or H) has not been found associated www.futuremedicine.com

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with PTEN deficiency in melanoma biopsies. However, it has to be noted that NRAS and PTEN deficiency were reported in melanoma cells in culture [38] . Efforts are currently being made to generate mice that accurately mimic human melanoma formation to allow further characterization of the effects of PTEN function and its role in the disease. One potential approach could be the generation of mice that can be conditionally and temporally repressed for PTEN at different periods of melanoma initiation and progression using, for instance, the tetracyclin system. The strict comparison of these different mouse melanoma models is difficult because they present many differences including the genetic background. This last parameter was shown to be very important for melanoma initiation [113] . Unfortunately, there are no available data concerning progression in which invasion and metastasis are involved. Conclusion

The available evidence suggests that PTEN exerts its tumor-suppressive effects via several mechanisms. Whether it is through senescence bypass, downregulating survival kinases or by interacting with chromatin, it is clear that PTEN is an important tumor suppressor in melanoma. Premalignant lesions contain PTEN but often also carry a hyperproliferative mutation in the form of BRAF V600E . The exact mechanism of tumorigenic induction is not known but the evidence implicating PTEN is increasing. As a therapeutic target, PTEN seems an unlikely candidate due to its propensity to be lost in malignant melanoma. However, developing strategies to target downstream components, such as mTOR, with small-molecule inhibitors may be an option to compensate for the loss of PTEN

function; such inhibitors, used in combination with other small-molecule inhibitors, may confer a survival advantage for patients. Further studies are needed to fully appreciate the role of PTEN in melanomagenesis and the importance of its numerous downstream effectors, which may or may not be post-translationally modified during the process. Work in this area should lead to an evaluation of the usefulness of some of these effectors both as biomarkers and therapeutic targets. Future perspective

The better knowledge of the structure and function of PTEN will allow discovery of its numerous interactors, and the functions of these various complexes. Such knowledge will lead to the better control of such interactions. Affecting these interactions will modulate the function of the specific complex. Another challenge will certainly be to understand the temporal importance of these different complexes. The temporal repression of PTEN at different periods of melanoma initiation and progression using, for instance, the tetracyclin system would certainly inform us on the role of this protein during melanomagenesis.u Financial & competing interests disclosure

A Conde-Perez was supported by a fellowship from Institut Curie. This work was supported by the Ligue Nationale Contre le Cancer (Equipe labellisée), Institut National sur le Cancer and Association de la Recherche sur le Cancer.�� The authors have no other relevant affili‑ �� ations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Executive summary Structure & function PTEN is a phosphatase (lipid and protein) primarily found in the cytoplasm, although it can also be found in the nucleus. PTEN regulates its own phosphatase activity by appropriate folding of the protein, when it is properly post-translationally modified. Molecular biology In melanoma, PTEN is found to be mutated (most frequently on exon 5) in 17% of melanoma cases. PTEN is deleted in 13% of melanoma cases. Human genetic diseases PTEN may be altered in various syndromes including Cowden’s, Lhermitte–Duclos and Bannayan–Zonana, resulting in increased susceptibility to developing breast and nonmedullary thyroid carcinoma, dysplastic gliocytoma and endometrial carcinoma, but not melanoma. PTEN mouse models Constitutive and conditional PTEN mutations have revealed the importance of this protein during homeostasis and melanomagenesis. The lack of PTEN in melanocyte stem cells renders them more resistant to hair graying. The presence of BRAF (V600E) as well as HRAS (G12V) mutations along with the absence of PTEN in melanocytes is sufficient to initiate melanomagenesis.

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nn

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